Considerable concerns and efforts have been directed to the development of new fetal hemoglobin (Hb F) inducers. Hydroxyurea (HU) is being used for treating sickle cell anemia (SCA), but the magnitude of response to HU significantly varies between SCA patients. The clinical efficacy of HU for beta-thalassemia remains unclear. Rather than studying cis-acting regulatory elements, our research has been focusing on clarifying intracellular pathways that regulate globin gene expression. We recently showed that an intracellular pathway comprising soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase (PKG) plays an important role in gamma-globin gene expression in primary erythroblasts. Our hypothesis that the sGC-PKG pathway plays an important role in Hb F expression was recently substantiated by a discovery of Italian brothers who have no mutations in beta-globin locus but express 25 to 30 % Hb F. They were found to have high levels of protoporphyrin IX, which is a strong activator of sGC. To study "with fresh eyes" the molecular mechanisms for gamma-globin gene silencing, we were concerned with a phenomenon that expression of the gamma- and beta-globin genes is regulated "in a reciprocal manner" at the perinatal period. This has led us to speculate that the gamma-globin gene is silenced in the adult stage by the mechanisms required for activating the beta-globin gene. We will test this hypothesis in this application. Our preliminary studies suggested that 1) cAMP-dependent protein kinase (PKA) activity is necessary for the induction of the beta-globin gene in adult erythroid cells such as MEL cells and primary erythroblasts, that 2) expression of the gamma-globin gene is decreased by activating cAMP-dependent pathways in K562 cells, and that 3) activity of HS2 and a gamma-globin gene promoter is markedly increased by suppressing PKA activity. These results suggest that cAMP-dependent pathways might play a negative role in gamma-globin gene expression. This application has three specific aims. In the first aim, we will characterize intracellular pathways that are required for activating beta-globin gene expression. The second aim will focus on the molecular mechanisms by which intracellular pathways for beta-globin gene expression silence the gamma-globin gene, In the third aim, we will examine whether expression of the gamma-globin gene is induced in primary erythroblasts of the beta-globin disorders and adult mice which have low activities of intracellular pathways for beta-globin gene expression. This application should not only shed much light on the mechanisms that silence the gamma-globin gene, but also provide a clue to the mechanisms underlying the developmental regulation of beta-like globin genes. Furthermore, important information to develop novel Hb F inducers will be disclosed by this study.